Harel Itamar, Valenzano Dario Riccardo, Brunet Anne
Department of Genetics, Stanford University, Stanford, California, USA.
Max Planck Institute for Biology of Ageing, Cologne, Germany.
Nat Protoc. 2016 Oct;11(10):2010-2028. doi: 10.1038/nprot.2016.103. Epub 2016 Sep 22.
A central challenge in experimental aging research is the lack of short-lived vertebrate models for genetic studies. Here we present a comprehensive protocol for efficient genome engineering in the African turquoise killifish (Nothobranchius furzeri), which is the shortest-lived vertebrate in captivity with a median life span of 4-6 months. By taking advantage of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system and the turquoise killifish genome, this platform enables the generation of knockout alleles via nonhomologous end joining (NHEJ) and knock-in alleles via homology-directed repair (HDR). We include guidelines for guide RNA (gRNA) target design, embryo injection and hatching, germ-line transmission and for minimizing off-target effects. We also provide strategies for Tol2-based transgenesis and large-scale husbandry conditions that are critical for success. Because of the fast life cycle of the turquoise killifish, stable lines can be generated as rapidly as 2-3 months, which is much faster than other fish models. This protocol provides powerful genetic tools for studying vertebrate aging and aging-related diseases.
实验性衰老研究中的一个核心挑战是缺乏用于基因研究的短寿命脊椎动物模型。在此，我们展示了一种在非洲青鳉（Nothobranchius furzeri）中进行高效基因组工程的综合方案，非洲青鳉是圈养条件下寿命最短的脊椎动物，平均寿命为4至6个月。通过利用成簇规律间隔短回文重复序列/CRISPR相关蛋白9核酸酶（CRISPR/Cas9）系统和青鳉基因组，该平台能够通过非同源末端连接（NHEJ）产生敲除等位基因，并通过同源定向修复（HDR）产生敲入等位基因。我们包括了针对向导RNA（gRNA）靶点设计、胚胎注射与孵化、种系传递以及最小化脱靶效应的指南。我们还提供了基于Tol2的转基因策略以及对成功至关重要的大规模饲养条件。由于青鳉的生命周期较短，稳定品系可在短短2至3个月内产生，这比其他鱼类模型要快得多。该方案为研究脊椎动物衰老及衰老相关疾病提供了强大的遗传工具。
